Azalide 3,6-Ketals: Antibacterial Activity and Structure–ActivityRelationships of Aryl and Hetero Aryl Substituted Analogues

Subas M. Sakya,* Peter Bertinato, Bryan Pratt, Melani Suarez-Contreras,Kristin M. Lundy, Martha L. Minich, Hengmiao Cheng, Carl B. Ziegler,

Barbara J. Kamicker, Shigeru F. Hayashi, Sheryl L. Santoro, David M. Georgeand Camilla D. Bertsche

Veterinary Medicine Research and Development, Pfizer Inc, Eastern Point Road, Box 4063, Groton, CT 06340, USA

Received 17 September 2002; accepted 16 December 2002

Abstract—Aryl and hetero aryl substituted 3,6-ketals of 15-membered azalide analogues were synthesized and were found to havepotent in vitro antibacterial activity against veterinary pathogens, including Staphylococcus aureus and Pasteurella multocida.# 2003 Elsevier Science Ltd. All rights reserved.

Mastitis is an inflammation of the mammary gland,usually caused by a variety of bacteria. One of the mostprevalent pathogens for non-lactating cow (dry cow)mastitis is Staphylococcus aureus. Mastitis is the mostimportant disease in the worldwide dairy industry andcauses considerable economic loss. The US NationalMastitis Council estimates that annual losses to thedairy industry amount to US$1.8–2 billion, or US$185–200 per cow. Yearly, between 30 and 50% of all dairycattle in the US are affected by mastitis.1,2

Antibiotic therapy is widely used to eliminate mastitis-causing infections and return udders to a healthy con-dition. These antibacterial agents include penicillins,tetracyclines, macrolides and a number of cephalospor-ins. Intramammary infusion (IMI) is commonly used todeliver the antibiotics. However, none of the treatmentsis significantly effective against mastitis caused by S.aureus. Here, we report our attempts to discover anti-bacterial agents with improved activity against S. aureuswhen administered by subcutaneous injection.

In the search for active compounds against bovinerespiratory disease (BRD), Lundy et al. discovered that3,6-ketals of the 9a-azalides showed excellent in vitroactivity against the Gram-negative pathogens, Pasteur-

ella multocida and Mannheimia (Pasteurella) haemoly-tica, responsible for BRD.3 These analogues,exemplified by 1, also showed potent in vivo activityagainst those pathogens.4 We initiated synthetic effortsto identify compounds with improved activity againstGram-positive mastitis pathogens using the 3,6-ketalazalide as a template. Recently we reported the SAR ofthe benzyl and aryl amide modifications on the 3,6-ketalnitrogen (Fig. 1) in which we showed improved activityby incorporation of heteroaryl moieties.4 In this paper,we will describe the results of the modification of thepiperidine ketal nitrogen with aryl and heteroarylderivatives.

0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0960-894X(03)00100-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1373–1375

Figure 1. Lead 3,6-piperidine ketal azalides.

*Corresponding author. Fax: +1-860-715-7312; e-mail: sakyas@groton.pfizer.com

Compound 6 was used as precursor material for syn-thesis of the 3,6-ketal analogues. The synthesis of 6 wascarried out using established procedures according toScheme 1.4

The syntheses of aryl piperidine ketal derivatives wereaccomplished by heating 3,6-ketal 6 with aryl fluoridesin the presence of base as shown in Scheme 2.

Scheme 3 shows the preparation of the 2-pyridyl, 2-pyrimidine, and 2-triazine derivatives. Generally mod-erate to good yields of the products were obtained.Similar reaction conditions were used for the prepara-tion of other heterocyles (8t and 8v–8y). The pyrimidinegroups were further functionalized to make several esterand amide derivatives.

While broad spectrum agents are desirable, activityagainst the Gram-positive bacteria S. aureus is essential.Among the substituted aryl analogues, 2-fluoro or 3-CF3-4-benzyloxycarbonyl substituted derivatives (7aand 7c) with 9a N-H showed potent activity against S.aureus (MIC: 0.39–0.78 mg/mL) but had less activityagainst the Gram-negative Escherichia coli. The corre-sponding 3-CF3-4-benzyloxycarbonyl aryl derivative(7b) with 9a N-Me showed improved activity against E.

coli. The nitrophenyl derivatives (7d and 7e) showedonly moderate activity against all three pathogens.Other functional groups containing aryl analogues didnot improve upon the activity against S. aureus.

Among the pyridine analogues, nitro- and cyano-sub-stituted analogues (8a–8c) showed good activity againstS. aureus and P. multocida, and reduced activity againstE. coli. Adding a more polar functionality to the mol-ecule (8d) improved the activity against E. coli. Thehydroxy methyl substituted analogue (8e) had the bestactivity against all three pathogens. Most analogues hadreasonable activity against all the pathogens with theexception of 8k (Table 1).

The pyrimidine analogues had the weakest activityagainst E. coli and P. multocida while having goodactivity against S. aureus. The more lipophilic 8l and8m had the best activity against S. aureus. Several ofthe pyrimidine analogues (8n, 8o, 8r, and 8s) hadmoderate activity against all three pathogens. Furthermodifications of the pyrimidine ring did not improveactivity.

The heterocyclic analogues 8s–8x did not show anyenhanced activity over the other analogues. Overall,very slight differences in the activity profile of thevarious aryl and heteroaryl derivatives of the 3,6-piperidine ketals were seen. A majority of the aryland heteroaryl analogues showed improved activityagainst S. aureus over the unsubstituted 3,6-ketal 6and reduced activity against E. coli. With the excep-tion of the rather lipophilic pyrimidine analogues,most analogues were active against P. multocida. Sev-eral of the active aryl, pyridine and pyrimidine analo-gues (7a, 7b, 7d, 7e, 8a, 8f, 8h, 8l, 8m, 8o, 8r, and 8u)were tested in a S. aureus murine intraperitoneal infec-tion model and showed poor in vivo activity (>20mg/kg ED50). Previous studies

5 had shown that polarity wasessential for improved in vivo activity for macrolides.Therefore, the lack of in vivo activity may be due tothe significantly increased lipophilicity of these newanalogues.

Aryl and heteroaryl linked piperidine 3,6-ketal azalideswith potent in vitro activity against both Gram-positiveand Gram-negative bacteria were discovered. There wasno significant difference in the in vitro activity against S.aureus between 9a N-H and N-Me analogues. Unlikethe previously reported compounds4 these lipophilicderivatives had minimal or no activity in a S. aureus invivo murine model.

In Vitro Assays

The microdilution method based on the NCCLS guide-lines6 has been reported previously.4 Minimum inhibi-tory concentration (MIC) was determined at the lowestdrug concentration that completely inhibits bacterialgrowth. The following three strains were used for theMIC determinations: 01A0758 (S. aureus), 51A0150 (E.coli), and 59A0067 (P. multocida).

Scheme 2. (a) CsCO3, aryl fluoride, base, 80–95�C, 60–85%.

Scheme 3. (a) Et3N, 2-chloro pyridine, 2-chloro pyrimidine, or 2-chloro triazine, CH3CN or iPrOH 80–95 �C, 60–85%.

Scheme 1. (a) pTsOH, 4, CH2Cl2, 60%; (b) H2, Pd/C, 99%.

1374 S. M. Sakya et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1373–1375

Murine Models

A S. aureus intraperitoneal infection model was descri-bed in a previous report.4 The number of surviving micewas counted after 4 days, and the effective dose for 50%survival (ED50) was calculated using a regressionequation.

Acknowledgements

We are grateful to Dr. Robert Rafka’s group for pro-viding us multi-gram quantities of 6 and Dr. John Dir-lam for many helpful discussions.

References and Notes

1. (a) Bramley, A. J.; Cullor, J. S.; Erskine, R. J.; Fox, L. K.;Harmon, R. J.; Hogan, J. S.; Nickerson, S. C.; Oliver, S. P.; Smith,L. K.; Sordillo, L. M. In Current Concepts of Bovine Mastitis; 4thed. National Mastitis Council, 1996; p 11. (b) Barkema, H. W.;Schukken, Y. H.; Lam, T. J. M.; Beiboer, M. L.; Wilmink, H.;Benedictus, G.; Brand, A. J. Dairy Sci. 1998, 81, 411.2. (a) Hogan, J. S.; Smith, K. L.; Hoblet, K. H.; Schoenber-

ger, P. S.; Todhunter, D. A.; Hueston, W. D.; Pritchard, D. E.;Bowman, G. L.; Heider, L. E.; Brockett, B. L.; Conrad, H. R.J. Dairy Sci. 1989, 72, 1547. (b) Clements, M. In AnimalPharm Reports, Bovine Mastitis: Products and Markets; PJB,Richmond, UK: June 30, 1998 p 9.3. (a) Minich, M. L.; Lundy, K. M. Abstract of Papers,Structural Elucidation of Descladinose Azithromycin-3,6-(Azacyclohexyl) Ketal by One- and Two-D NMR Spec-troscopy, 218th National Meeting of the American Che-mical Society, New Orleans, LA, Aug 22–26, 1999. (b)Minich, M. L.; Lundy, K. M.; Rafka, R. J.; Morton, B. J.Abstract of Papers, Azalide 3,6-Ketals: Discovery of a NovelClass of Azalide Antibiotics, 218th National Meeting of theAmerican Chemical Society, New Orleans, LA, Aug 22–26,1999.4. Cheng, H.; Dirlam, J. P.; Ziegler, C. B.; Lundy, K. M.;Hayashi, S. F.; Kamicker, B. J.; Dutra, J. K.; Daniel, K. L.;Santoro, S. L.; George, D. M.; Bertsche, C. D.; Sakya, S. M.;Suarez-Contreras, M. Bioorg. Med. Chem. Lett. 2002, 12,2431.5. McFarland, J. W.; Berger, C. M.; Froshauer, S. A.; Haya-shi, A. F.; Hecker, S. J.; Jaynes, B. H.; Jefson, M. R.;Kamicker, B. J.; Lipinski, C. A.; Lundy, K. M.; Reese, C. P.;Vu, C. B. J. Med. Chem. 1997, 40, 1340.6. Performance standards for antimicrobial disk and dilutionsusceptibility tests for bacteria isolated from animals:Approved standard, M31-A, Vol. 19, No. 11, June, 1999.

Table 1. Biological data for aryl and hetero aryl piperidine amine-3,6-ketals

Compd

Ar MIC (mg/mL)

R (9a)

S. aureus E. coli P. multocida ClogP

6

N/A Me 6.25 0.39 <0.05 1.454 7a 2-Fluoro-4-benzyloxycarbonyl phenyl H 0.39 >50 3.13 5.529 7b 3-CF3-4-Benzyloxycarbonyl phenyl Me 0.78 3.13 0.39 7.61 7c 3-CF3-4-Benzyloxycarbonyl phenyl H 0.78 >50 6.25 6.757 7d 2-Nitro phenyl Me 1.56 3.13 0.39 4.698 7e 4-Nitro phenyl Me 1.56 6.25 0.39 4.698 7f 2-Fluoro-4-pyridyl methyl amido phenyl Me 3.13 1.56 0.2 3.754 7g 4-Cyano phenyl Me 3.13 25 0.39 4.419 7h 3-CF3-4-COOH-phenyl Me 25 12.5 3.13 2.554 7i 4-Benzyloxycarbonyl phenyl Me 3.13 3.13 0.39 6.638 7j 4-(4-F-Phenylsulfonyl) phenyl Me 3.13 >50 1.56 5.573 7k 4-Benzoylamino phenyl Me 12.5 50 1.56 5.097 7l 4-Amino phenyl Me 6.35 12.5 1.56 3.362 8a 3-Nitro-2-pyridyl H 0.78 6.25 0.20 2.695 8b 3-Cyano-6-Me-2-pyridyl H 0.78 6.25 0.78 2.898 8c 4-Methyl-5-nitro-2- pyridyl H 0.78 6.25 1.56 3.114 8d 5-Nitro-6-amino-2-pyridyl H 1.56 3.13 0.1 2.974 8e 4-Hydroxymethyl-6-chloro-2-pyridyl H 1.56 0.78 0.2 2.49 8f 5-Cyano-2-pyridyl H 1.56 6.25 0.20 2.399 8g 5-Amido-2-pyridyl H 1.56 6.25 0.20 1.929 8h 5-Nitro-2-pyridyl H 1.56 3.13 0.39 2.695 8i 5-CF3-2-pyridyl H 1.56 12.5 0.78 3.805 8j 5-Nitro-2-pyridyl Me 3.13 6.25 0.39 3.548 8k 5-Ethoxycarbonyl 2-pyridyl H 6.25 50 3.13 3.426 8l 4-CF3-5-Benyloxycarbonyl-2-pyrimidine H 0.78 >50 >50 4.675 8m 4-CF3-5-Benyloxycarbonyl-2-pyrimidine Me 1.17 >50 >50 5.528 8n 2-Pyrimidine Me 1.56 1.56 0.10 2.849 8o 4-CF3-2-Pyrimidine H 1.56 3.13 0.78 2.923 8p 5-(Pyrid-4-ylmethyl amido)-2-pyrimidine Me 3.52 >50 >50 2.408 8q 4-CF3-2-Pyrimidine Me 6.25 12.5 1.56 3.776 8r 2,4-Methoxy-6-pyrimidine H 1.56 3.13 0.2 3.72 8s 2,4-Methoxy-6-pyrimidine Me 1.56 6.25 0.39 4.576 8t 6-Chloro-2-pyrazine Me 1.56 6.25 0.39 3.537 8u 4-Chloro-6-phenyl-2-1,3,5-triazine Me 1.56 >50 >50 4.63 8v 2-Benzimidazole Me 3.13 1.56 0.1 4.561 8w 2-Thiazole Me 3.13 3.13 0.39 3.519 8x 2-Benzoxazole Me 3.13 6.25 0.78 4.411 8y 6-Methyl-3-pyridazine Me 6.25 3.13 0.20 3.183

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